ARTICLE pubs.acs.org/JAFC
Reliable Enzyme-Linked Immunosorbent Assay for the Determination of Coconut Milk Proteins in Processed Foods Vipa Surojanametakul,*,† Hirotoshi Doi,‡ Haruki Shibata,‡ Tasuku Mizumura,‡ Toshio Takahashi,‡ Warunee Varanyanond,† Sirinrat Wannapinpong,† Masahiro Shoji,‡ Tatsuhiko Ito,‡ and Hirotoshi Tamura§ †
Institute of Food Research and Product Development (IFRPD), Kasetsart University, 10900, Bangkok, Thailand Morinaga Institute of Biological Science Inc., Kanazawa-ku, Yokohama, Japan § Graduate School of Agricultural Science, Kagawa University, Kagawa, Japan ‡
ABSTRACT: This study was designed to develop a novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of coconut milk proteins in processed foods. The developed sandwich ELISA was able to detect coconut milk proteins from various coconut milk products and did not show any cross-reactivity with 41 of 42 kinds of popularly used food ingredients, thus reflecting great specificity for coconut milk proteins. In addition, the established ELISA is highly sensitive and allowed the detection of 0.31 μg/g of coconut milk protein in complex food matrices. This proposed assay could serve as a useful tool for the detection of the presence of hidden coconut milk proteins in processed foods. KEYWORDS: coconut milk, food allergy, sandwich ELISA
’ INTRODUCTION Reactions triggered by food allergens are increasing and becoming a major health concern worldwide, especially in industrialized countries. It is estimated that up to 8% of children and 2% of adults are affected by food allergies,1-3 with symptoms ranging from relatively mild to severe anaphylactic reactions with fatal consequences.4 Many foods are known to trigger food allergies; among these, eight types (peanuts, nuts, wheat, soy, milk, eggs, fish, and shellfish) are responsible for causing the majority (>90%) of allergic reactions.5 Tree nuts are one of the most potent of all known food allergens and are often attributed as the cause of food anaphylaxis and anaphylactic death.6-8 Among tree nuts, those most commonly responsible for allergic reactions are hazelnut, walnut, cashew, and almond, whereas those less frequently associated with allergies are pecan, chestnut, Brazil nut, pine nut, macadamia nut, pistachio, and coconut.8 Although technically not a tree nut, coconut has been categorized as such by the U.S. Food and Drug Administration (FDA) since 2006. Allergy to coconut is very infrequent.9,10 However, coconut can elicit allergic reactions, including immediate reaction and anaphylaxis, especially in patients who have been previously diagnosed with a severe sensitivity to nuts.9,11-13 Cross-reactivity between coconut and other tree nuts, such as hazelnuts and walnuts, has been demonstrated.12,13 This probably increases the risk of coconut allergy in consumers who are allergic to tree nuts or peanuts, although there is one study that showed no evidence.14 Since the FDA updated their tree nut allergen list to include coconut, foods that contain coconut ingredients have required mandatory labeling. Coconut (Cocos nucifera) is one of the most economically important palm species. Its endosperm, particularly in the form of coconut milk, is widely used as an ingredient in many Asian cuisines, including Thailand.15 Food products containing r 2011 American Chemical Society
coconut include curry, biscuits, dessert mix, and ice cream. In most factories, many different food products are produced, with various food ingredient compositions. A problem of hidden allergens might result from cross-contamination in the food production line, through inadequate cleaning of shared equipment or by reworking of allergen-containing products.16,17 Thus, strict control of coconut allergens in food factories is required to minimize the risk to coconut-allergic consumers and to comply with product labeling regulations. To practically and effectively manage food and food ingredients, especially traces of coconut, a simple and reliable detection method for detecting hidden coconut allergens is necessary. Presently, numerous ELISA kits suitable for specific nut allergen detection (hazelnuts, almonds, walnuts, etc.) are available,7,18,19 whereas only one commercial product offers a lateral flow test for the qualitative detection of coconut allergen. To date, no quantitative enzyme-linked immunosorbent assay (ELISA) test suitable for coconut milk allergen determination has been developed. Coconut allergens are identified to be seed storage proteins such as 11S (legumin-like) globulin, known as cocosin, and 7S (vicilinlike) globulin.11 Globulins comprise 75% of the total protein in coconut; of this, 11S is estimated to constitute 86% and 7S, 14%.20 Some researchers have already shown that cocosin, a major coconut allergen, is a hexamer with a molecular mass of approximately 300 kDa, with each heterogeneous 54 kDa subunit comprising an acidic and basic polypeptide chain linked by disulfide bonds.11 Under reducing conditions, these proteins migrate on gel electrophoresis as bands of about 35 and 32 kDa and a complex of 22 kDa. The 7S globulin, on the other hand, is a trimeric protein with a native Received: April 9, 2010 Revised: January 14, 2011 Accepted: January 31, 2011 Published: February 17, 2011 2131
dx.doi.org/10.1021/jf104067v | J. Agric. Food Chem. 2011, 59, 2131–2136
Journal of Agricultural and Food Chemistry
Figure 1. SDS-PAGE of defatted coconut milk IV, P40-60, defatted hazelnut, and defatted walnut. Lanes: (1) molecular weight marker; (2) defatted walnut; (3) defatted hazelnut; (4) defatted coconut milk IV; (5) P40-60. Each protein sample (2 μg) was separately applied to lanes 2, 3, 4, and 5. Arrows indicate molecular weight markers.
molecular mass of ∼150 kDa. It shows no disulfide bond20 and is quickly digested by pepsin.11 However, during the processing of coconut milk proteins, the reactivity of the allergenic proteins might be altered due to structural changes, affecting their ability to be detected in the food matrix. Thus, it is very important to obtain the specific antibodies that can recognize denatured coconut milk proteins for ELISA in certain foods after processing. Preparation of these antibodies presents a challenge. Moreover, a major problem with most ELISAs developed for allergen detection from nuts and seeds is that many nuts and seeds are closely related phylogenetically.21 Therefore, a highly sensitive and specific ELISA without cross-reactivity is crucial, to avoid incorrect judgments. The aim of this study was to develop the first highly coconut specific and sensitive sandwich ELISA that can detect and quantify trace amounts coconut milk proteins in processed foods. This assay is developed to be used by the food industry and regulatory agencies to protect consumers and to avoid loss of sales due to allergenrelated recalls.
’ MATERIALS AND METHODS Food Materials. Three kinds of coconut milk and dried coconut were purchased at local supermarkets (Bangkok, Thailand) in 2009. Coconut milk I is 100% coconut milk, Chaokoh brand, made in Thailand (Ampol Food Processing Co., Ltd.). Coconut milk II is 100% coconut milk, Aroy-D brand, made in Thailand (Thai Agri Foods Public Co., Ltd.). Coconut milk III is 100% coconut milk scented candles, Chaokoh brand, made in Thailand (Ampol Food Processing Co., Ltd.). Dried coconut meat was prepared from fully mature coconut endosperm and cut into small pieces before drying at 80 °C in a hot-air oven for 2 h. Two other kinds of coconut milk were purchased at local supermarkets in Yokohama, Japan, in 2009. Coconut milk IV was canned 100% coconut milk, Ayam brand, made in Malaysia. Coconut milk V was a canned
ARTICLE
Figure 2. Western blotting analysis of defatted coconut milk, P40-60, defatted walnut, and defatted hazelnut. Lanes: (1) molecular weight marker; (2) defatted walnut; (3) defatted hazelnut; (4) defatted coconut milk IV; (5) P40-60. Each protein sample (0.1 μg) was separately applied to lanes 2, 3, 4, and 5. Arrows indicate molecular weight markers. product made of coconut milk (53%), water, citric acid, and sulfite (for bleaching), Chaokoh brand, made in Thailand (Thep Padung Porn Coconut Co., Ltd.). Chemicals and Reagents. 2-Mercaptoethanol (2-ME), sodium sulfite, sodium dodecyl sulfate (SDS), polyoxyethylene sorbitan monolaurate (Tween 20), ammonium sulfate, and hexane were supplied by Nacalai Tesque, Inc. (Kyoto, Japan). Albumin from bovine serum (BSA), horseradish peroxidase (HRP), and 3,30 5,50 -tetramethylbenzidine (TMB) were provided by Wako Pure Chemical Industries, Co., Ltd. (Osaka, Japan), Toyobo Co., Ltd. (Osaka, Japan), and SurModics, Inc. (Eden Prairie, MN), respectively. In addition, the following buffers were prepared for the experiment: 150 mM Tris-HCl (pH 7.4) containing 0.1% (w/v) BSA and 0.05% (v/v) Tween 20 (buffer A); and 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl and 0.05% (v/v) Tween 20 (buffer B). Preparation of Coconut Milk Protein (P40-60). Coconut milk IV was defatted with hexane (1:2.5 v/v) by stirring for 2 h; then the hexane layer was removed after centrifugation at room temperature (5000g, 5 min). The procedure of defatting was repeated two times. Two hundred and twenty milliliters of defatted coconut milk was obtained from 400 g of coconut milk. Defatted coconut milk was added to solid ammonium sulfate at 40% saturation with stirring for 30 min at 4 °C followed by centrifugation (15000g, 30 min, 4 °C). The supernatant was then added to solid ammonium sulfate at 60% saturation under the same conditions. The precipitated proteins were centrifuged (15000g, 30 min, 4 °C) and dissolved in 20 mM Tris-HCl (pH 7.4) buffer. The precipitated proteins (P40-60) were used as antigens. Preparation of the Antibody to P40-60. The rabbit antiserum against P40-60 was produced by immunization of Japanese white rabbits using P40-60 solution (1 mg for P40-60, PBS containing 0.6% SDS (w/v), 0.1 M sodium sulfite) in Freund’s complete adjuvant. Injections were repeated five times at the appropriate intervals (14 days). Whole blood was collected, and the serum was separated. Specific polyclonal antibodies against denatured P40-60 were purified from the serum using a 1 mL HiTrap NHS-activated column (GE Healthcare, U.K.) in which 10 mg of the denatured coconut protein from dried coconut was fixed. The obtained antibodies were examined for the construction of the ELISA for detection of the coconut milk protein. 2132
dx.doi.org/10.1021/jf104067v |J. Agric. Food Chem. 2011, 59, 2131–2136
Journal of Agricultural and Food Chemistry
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SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Samples were diluted 1:1 with sample buffer (4% w/v SDS, 28% v/v glycerol, 0.24 mg/mL bromophenol blue, 20% v/v 2-ME). Under reducing conditions, the samples were boiled for 5 min. Polyacrylamide gels and molecular weight standards used were 12.5% Q-PAGE mini (TEFCO, Tokyo, Japan) and SeeBlue Plus2 Prestained Standard (Invitrogen Life Technologies Corp., Carlsbad, CA). Electrophoresis was run at constant voltage (200 V) for 36 min using a STC-808 (TEFCO). The gels were stained with Rapid CBB KANTO (Kanto Chemical Co., Inc., Tokyo, Japan). Western Blotting. Samples were subjected to SDS-PAGE and transferred onto a PVDF membrane (Amersham Hybond-P, GE Healthcare, U.K.) by a Trans-Blot SD semidry transfer cell (Bio-Rad Laboratories, Inc., Hercules, CA). The PVDF membrane was incubated for 1 h in a blocking solution (buffer B containing 0.1% BSA). Coconut protein was detected on blot using subsequently anti-P40-60 antibodies (20 ng/mL, 60 min), biotinylated anti-rabbit IgG antibodies (1 μg/mL, 30 min) from a Vectastain ABC-AP Kit (Vector Laboratories, Inc., Burlingame, CA), and alkaline phosphatase conjugated streptavidin (20 min) also from a Vectastain ABC-AP Kit, and it was used according to the manufacturer’s description. Signals were detected by a BCIP/NBT Alkaline Phosphatase Substrate Kit IV (Vector Laboratories, Inc.). Sample Extraction. A Millser IFN-700G homogenizer (Iwatani International Corp., Osaka, Japan) was used to homogenize the food samples. Samples were treated with the Millser a few times for 30 s until homogenized. Nineteen milliliters of buffer A, containing additional 0.6% (w/v) SDS and 0.1 M sodium sulfite, was added to 1 g of a homogenized sample. The sample was then shaken at 90-110 rpm over 12 h at 25 °C for extraction. After confirmation of sample pH at around 6.0-8.0 with pH paper, the sample was centrifuged at 3000g for 20 min;
Figure 3. Representative calibration curve of the developed ELISA. The calibration curve shows the mean value of 10 experiments and the standard deviation in each point of the curve. Concentrations of standard solution: 0.78 ng/mL (0.31 μg/g), 1.56 ng/mL (0.62 μg/g), 3.13 ng/mL (1.25 μg/g), 6.25 ng/mL (2.5 μg/g), 12.5 ng/mL (5 μg/g), 25 ng/mL (10 μg/g), and 50 ng/mL (20 μg/g).
the supernatant was then filtered through 5A filter paper (Advantec Toyo Kaisha, Ltd., Tokyo, Japan) to obtain the extract. As needed, the obtained extracts were analyzed with a 2-D Quant protein assay kit (GE Healthcare, U.K.). The sample extract, diluted 20-fold with buffer A, was used for ELISA. In the case of additional dilution, the 20-fold diluted sample extract was diluted with buffer A containing 0.03% (w/v) SDS and 0.005 M sodium sulfite. Preparation of Calibration Standard Solution. For the determination of coconut milk protein using ELISA, the preparation of a calibration standard solution is necessary. Coconut milk I powder, after freeze-drying, was defatted with hexane (1:2 w/v) by stirring for 1 h; this was followed by paper filtration. The procedure of defatting was repeated three times. Twenty milliliters of buffer A containing 0.6% (w/ v) SDS and 0.1 M sodium sulfite was added to 0.2 g of the defatted coconut milk powder. The mixture was then shaken at 90-110 rpm for 16 h at 25 °C for extraction. The extract was centrifuged at 10000g for 30 min, and the supernatant was filtered through a 0.8 μm microfilter (DISMIC-25CS, Toyo Roshi Kaisha, Ltd., Tokyo, Japan). The protein content of the filtrate was assayed using a 2D Quant protein assay kit (GE Healthcare, U.K.). The original filtrate was diluted to 10 times its volume with phosphate buffered saline (PBS, pH 7.4). Next it was further diluted to twice its volume by PBS (pH 7.4) containing 0.2% BSA and then stored at -80 °C for preservation. Finally, it was diluted to 50 ng/mL using buffer A containing 0.03% (w/v) SDS and 0.005 M sodium sulfite. The prepared calibration standard solution for the ELISA was stored at 4 °C. ELISA. A microtiter plate (F8Maxisorp Nunc-Immuno module, Thermo Fisher Scientific Inc., Waltham, MA) was coated at 25 °C for 2 h with the prepared specific polyclonal antibody to anti-P40-60 (100 μL of 2.5 μg/mL antibody protein solution in 50 mM sodium carbonate, pH 9.6). The plate was blocked at 25 °C for 2 h with buffer B containing 0.1% w/v BSA, as described in a previous study.7 The blocking buffer was removed, followed by drying of the plate. The diluted food sample and the coconut milk standard solution were added to the plate (100 μL/ well) and incubated for 1 h at 25 °C. After six washings with buffer B, the anti-P40-60 antibody labeled with HRP using the method described in previous literature22 was diluted with buffer B containing 1% BSA. This was added to the ELISA plate at 100 μL/well and allowed to stand for 30 min. After six washings with buffer B, TMB (100 μL/well) was added, and the enzyme reaction was allowed to run at 25 °C for exactly 10 min. The reaction was stopped by the addition of 100 μL/well of 0.5 M H2SO4. The absorbance was measured at 450 nm, the dominant wavelength, and at 620 nm, the subdominant wavelength. The amount of coconut milk protein was calculated using the calibration standard curve of coconut milk protein based on P40-60 assay as a coconut marker protein. All experiments were performed in duplicate. Each sample determination was expected to have a mean CV of
